There has been a trend in recent years toward miniaturization of laboratory tests involving chemical reactions in order to conserve expensive reagents and limited quantities of samples to be analyzed, and to facilitate mechanical automation, as set forth by applicant in "Semiautomation of Immunoassays by Use of Magnetic Transfer Devices", Methods in Enzymology, Volume 70, pages 388-416, 1980. Each of the reaction wells in a typical 96-well tray used in serologic work can contain a maximum of approximately 300 microliters; the volume of reactants actually dispensed in each such well normally ranges from 100-200 microliters. For this reason, precision made microliter pipettes or other small-volume liquid transfer devices are used for handling and diluting the reactants.
In cases where samples need to be initially diluted minimally, e.g. 1:2-1:20, several volumetric liquid transfer devices have been used. U.S. Pat. No. 3,252,331 teaches apparatus which accomplishes serial dilution of a sample by fluid trapped in said apparatus and, after mixing in a receptacle containing a diluent fluid, a fixed volume of the diluted material can be transferred to another receptacle, mixed, and such steps repeated.
In cases where samples must be substantially diluted (as for example 1:50 or more) before being tested in one of these miniature analytical systems, the dilutions are usually made in intermediate, larger vessels, then small volumes of the diluted materials are transferred to the miniature reaction wells in which the test is to be conducted.
Heretofore there has been no satisfactory way to make accurate-precise dilutions in small volumes, principally of ten microliters or less, directly into miniature reaction wells. This problem can be readily understood, for example, when one considers the need to make, in one step, a 1:50 initial dilution of a serum sample so that the total volume will be only 100-200 microliters; the volume of serum sample required is either 2 or 4 microliters, volumes not conveniently or easily measured accurately-precisely with existing equipment. A presently used alternative, as heretofore mentioned, is to use existing equipment to make the initial dilution in an intermediate vessel in suitably large volumes, then transfer a small volume of the diluted sample into the miniature reaction well for testing. This obviously requires an extra step in the assay process, necessitates the use of one or more extra intermediate vessels, extra pipettes and/or pipette tips, and additional time is required, all of which invites human errors inevitably associated with multiple manipulations.